Use of flow-cytrometric analysis to optimize cell banking strategies for cho cells

ABSTRACT

Production of biopharmaceuticals from CHO cells requires generation of master-, working- and post-production cell banks of high quality, partly under GMP conditions. An optimal cryopreservation strategy is needed for each new production cell line, particularly with regard to the desire to establish production processes that are completely devoid of serum or even any animal components and to ensure robust thaw performance for reliable production. Here we describe a novel strategy employing flow cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of CHO cell banks. Our data show that this method enables evaluation of a cryopreservation procedure just 6 h after thaw.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the production of biopharmaceuticals inCHO cells. Particularly, it pertains to the generation of master-,working- and post-production cell banks of high quality viacryopreservation. More particularly, it pertains to the propagation andcharacterization of cells cryopreserved in master-, working- andpost-production cell banks. Furthermore, the present invention refers toa novel strategy employing flow cytometric (FC) analysis of AnnexinV-stained cells for high-throughput characterization of cryopreservedcell banks.

2. Background of the Invention

The market for biopharmaceuticals for use in human therapy continues togrow at a very high rate in the last decade. CHO cell lines are one ofthe most attractive mammalian expression system for production, safety,and regulatory aspects. To ensure therapeutic products of uniformquality, the cell banking system of these cell lines is crucial.Creation of Master Cell Banks (MCB), Working Cell Banks (WCB), and PostProduction Cell Banks (PPCB) of CHO cells are essential steps indevelopment of production processes for biopharmaceuticals in that celllines. The quality of these banks is critical, as their generation notonly supports clinical development of the product but also ultimatelythe market supply phase.

The main parameter that characterizes the quality of a cell bank is thelong term survival of cultured cells after thawing. Moreover, besidesthe long term survival, robustness and stability are also essentialproperties of a suitable cell bank. The time it takes from thawing avial to establishing inoculum cultures of robust growth, geneticstability and high culture viability is critical for assessing thequality of a cell bank. Finally, a cell bank of high quality shouldguarantee for all of these parameters to remain stable over a prolongedstorage period of the bank. All these characteristics highly depend onthe method of cryopreservation for a given production cell line.

Today, an increasing number of biopharmaceuticals is produced from CHOcells due to their ability to correctly process and modify humanproteins. The first generation of CHO cell-based production processesalmost exclusively required the presence of serum in the culture medium.Safety and regulatory benefits led to development of new cell lines andculture regimes that now enable serum-free cultivation of cellsthroughout the process (Merten, 1999). However, the removal of serumfrom the entire production process also requires cells to be stored inmaster and working cell banks with serum-free freezing media. A varietyof strategies have been described for cell banking of cells by usingcryoprotectants that are able to at least partially replace theprotective effects of serum (Groth et al., 1991). However, the successof any such strategy highly depends on the cell line, the medium, andthe protocol for freezing and thawing. Therefore, evaluation ofdifferent cryopreservation strategies is essential for successfulprocess development.

Currently, the first assessment of a newly generated cell bank isperformed by thawing a defined number of vials and culturing cells for5-10 passages. Cell number and viability as determined by trypan blueexclusion are the routinely used parameters to describe the recovery ofcells after cryopreservation.

Programmed cell death or apoptosis is a process crucial for properembryonic development and tissue homeostasis in the adult. Programmedcell death is controlled by a specific subset of molecules conserved inall multicellular organisms that converts a death inducing signal intointracellular biochemical processes, which ultimately lead to thecomplete destruction of the cell (Vaux and Korsmeyer 1999). Oncetriggered, apoptosis proceeds, with different kinetics depending on celltypes, and culminates with cell disruption and formation of apoptoticbodies. A critical stage of apoptosis involves the acquisition ofsurface changes by dying cells that eventually results in therecognition and the uptake of these cells by phagocytes. Differentchanges on the surface of apoptotic cells such as the expression ofthrombospondin binding sites, loss of sialic acid residues and exposureof phospholipids, like phosphatidylserine (PS), were previouslydescribed. Phospholipids are asymmetrically distributed between innerand outer leaflets of the plasma membrane, with phosphatidylcholine andsphingomyelin exposed on the external leaflet of the lipid bilayer andphosphatidylserine predominantly observed on the inner surface facingthe cytosol. Cells undergoing apoptosis break up the phospholipidasymmetry of their plasma membrane and expose PS, which is translocatedto the outer layer of the membrane. This occurs in the early phases ofapoptotic cell death during which the cell membrane remains intact. PSexposure is, thus, an early and wide-spread hallmark of dying cells.Annexin V, belonging to a recently discovered family of proteins, theannexins, with anticoagulant properties, has proven to be a useful toolin detecting apoptotic cells, since it preferentially binds tonegatively charged phospholipids, like PS, in the presence of Ca²⁺ andshows minimal binding to phosphatidylcholine and sphingomyelin. Changesin PS asymmetry analyzed by measuring Annexin V binding to the cellmembrane were detected before morphological changes associated withapoptosis occurred and before membrane integrity was lost.

By conjugating FITC to Annexin V it is possible to identify and quantifyapoptotic cells on a single-cell basis by flow cytometry (Steensma etal., 2003). Simultaneous staining of cells with FITC-Annexin V (greenfluorescence) and the non-vital dye propidium iodide (red fluorescence)allows (bivariant analysis) the discrimination of intact cells(FITC−PH−), early apoptotic (FITC+PI−) and late apoptotic or necroticcells (FITC+PI+).

SUMMARY OF THE INVENTION

As mention in the background section, quality of a cryopreserved cellbank is critical in the use of a cell bank for the production ofbiopharmaceuticals. In the meaning of this invention, quality means,post thaw vitality, robustness, phenotypic/genetic stability, and longterm preservation quality of cells, propagated and expanded startingfrom a cryopreserved cell bank. The present invention is based on thesurprising observation that programmed cell death, particularlyapoptosis, is the major cause of cell death after thawing ofcryopreserved CHO cells of a frozen CHO cell bank. It also has beensurprisingly demonstrated that the evidence of early apoptosis in cellsof a culture propagated and expanded from a cryopreserved cell bank ofCHO cells correlates with the quality of the CHO cell bank, which meanswith post-thaw vitality, robustness, phenotypic/genetic stability, andlong term preservation quality, observed when cells are propagated andexpanded starting from this cell bank. It has been furthermoredemonstrated that Annexin V is a suitable marker to detect earlyapoptosis in CHO cells that have been thawed after cryopreservation.

The present invention therefore pertains to the use of Annexin V incharacterizing a cryopreserved cell bank of CHO cells. Particularly, thepresent invention relates to the use of Annexin V in a process ofcharacterizing a cryopreserved cell bank of CHO cells, preferablyshortly after thawing a portion of cells of such a cell bank. Accordingto a further embodiment of the present invention, said process includesthe steps: a) thawing a portion of cells of a cryopreserved cell bank;b) cultivating said cells in a culture medium; c) incubating said cellswith Annexin V; d) detecting cells and quantifying the number of cellsthat binds to Annexin V; e) detecting cells and quantifying the numberof cells that do not bind to Annexin V; f) calculating the ratio ofAnnexin V-binding versus Annexin V-non-binding cells.

According to a further embodiment, the present invention also pertainsto the use of Annexin V in a process of determining the quality of a CHOcell bank, wherein said process includes the steps: a) cryopreservingcells into parts in a liquid medium as a cell bank; b) thawing a portionof the cryopreserved cells of said cell bank; c) cultivating said cellsin a culture medium; d) establishing the vitality-rate of said thawedcells by staining said cells with Annexin V. “Cryopreserving cells intoparts” in a liquid medium means, that each about 0.25 to 3E7 cells arefrozen in one to two ml of a liquid medium in a container. Per cellbank, about 200 containers or vials are frozen.

The present invention also pertains to the use of a Kit comprisingAnnexin V for the characterization of a cryopreserved cell bank of CHOcells post thawing, wherein Annexin V is used to determine thevitality-rate of the cryopreserved cells post thawing. According to apreferred embodiment of the present invention, the vitality-rate ofcryopreserved cells post thawing is determined by a process includingthe steps a) thawing a portion of cells of a cryopreserved cell bank; b)cultivating said cells in a culture medium; c) incubating said cellswith Annexin V; d) detecting cells and quantifying the number of cellsthat binds to Annexin V; e) detecting cells and quantifying the numberof cells that do not bind to Annexin V; f) calculating the ratio ofAnnexin V-binding versus Annexin V-non-binding cells. Furthermore, thepresent invention also provides a Kit comprising Annexin V and a packageleaflet including the information to use Annexin V in characterizationof a cell bank of CHO cells by the inventive process, described herein.

The present invention further provides a process of characterizing a CHOcell bank including the steps a) thawing a portion of cells of acryopreserved cell bank; b) cultivating said cells in an appropriateculture medium; c) incubating said cells with Annexin V; d) detectingcells and quantifying the number of cells that binds to Annexin V; e)detecting cells and quantifying the number of cells that do not bind toAnnexin V; f) calculating the ratio of Annexin V-binding versus AnnexinV-non-binding cells.

According to a further embodiment, the present invention also provides aprocess of measuring the vitality-rate of a CHO cell bank post-thawing,including the steps a) thawing a portion of cells of a cryopreservedcell bank b) cultivating said cells in an appropriate culture medium c)incubating said cells with Annexin V; d) detecting cells and quantifyingthe number of cells that binds to Annexin V; e) detecting cells andquantifying the number of cells that do not bind to Annexin V; f)calculating the ratio of Annexin V-binding versus Annexin V-non-bindingcells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Fingerprint of cell death of CHO cell culture postcryopreservation. CHO DG44 cells were frozen using a freezing machine ata cell concentration of 2E7 cells/mL in 90% cultivation medium and 10%DMSO. Cells were analyzed by FACS at indicated timepoints. The firstlane depicts forward scatter on the Y axis and side scatter on the Xaxis. Gates were set to distinguish cells that represent dead or livingcells as judged by their morphology. Lane 2 shows the same graphs andAnnexin V positive CHO cells are shown in white. Lane 3 depictspropidium iodide (PI) staining plotted versus Annexin V staining. Dotsare grey according to the forward versus side scatter gating.

FIG. 2 shows 6 h post-thaw Annexin V staining predicts cell bankquality. A. CHO DG44 cells were frozen using a freezing machine at acell concentration of 2E7 cells/mL in 90% cultivation medium and 10%DMSO. The size of the cell bank was 220 vials. A) FACS analysis ofAnnexin V stained cells 6 h post thaw. Data are plotted as forward sidescatter graph (gates as described for FIG. 1) and as Annexin V intensityplot. B) Growth and viability of post-thaw cultures. Cells were countedfor every passage. Simultaneously, viability was determined by trypanblue exclusion.

FIG. 3 shows different cell banks show different apoptosis after thaw.CHO DG44 was frozen using a freezing machine with a cell concentrationof 1E7 or 2E7 cells/mL in different freezing media. The size of the cellbanks was >200 vials. Cells were cryopreserved using a freezing machineor a polystyrene box in two different freezing media. Cultures wereanalyzed by Annexin V staining 6 h post thaw.

FIG. 4 shows use of early apoptosis measurements for testing of mediumquality. CHO DG44 cells were frozen using a freezing machine at a cellconc. of 2E7 cells/mL in 90% cultivation medium and 10% DMSO. The sizeof the cell bank was 220 vials. Different media preparations were usedfor generation of three cell banks. Cultures were analyzed by FACS 6 hpost thaw for cell cycle distribution, forward-side scatter, and AnnexinV staining.

DETAILED DESCRIPTION OF THE INVENTION

The term “cell culture(s) was (were) initiated” refers to the timepoint, when cryopreserved cells are thawed and transferred to cultureconditions suitable for the propagation and grown up of said cells to acell culture.

The term “cell culture” means multiple cells cultivated in one containerunder conditions suitable for the growth of the cells.

The term “post thaw vitality” shall mean the time-course of post thawrecovery of cells propagated from a given cell bank measured in thepercentage of viable cell to non-viable cells at the time ofsub-cultivation. This parameter is essential for the time it takes toestablish robust inoculum cultures and subsequent scale up for largescale cultivation. It therefore is a critical process parameter,

The term “robustness” describes the reproducibility with which cellcultures recover after cryopreservation according to an established postthaw vitality pattern. A robust cell bank would result in minordifferences amongst the post thaw performances of its different vials.The term “vial/vials” means a number of cells frozen in one container.Normally, a vial contains about 0.25 to 3E7 cells in one to two ml of aliquid medium.

The term “phenotypic/genetic stability” of cells is defined by thechange in RNA and protein expression level observed when cells from agiven cell bank are cultivated over a period of time relevant to thegiven process format, e.g., for about 100 to 300 sub-passages. A cellbank of poor quality, meaning low post-thaw vitalities could give riseto poor phenotypic/genetic stability due to a high selection pressurewhen cultures are initiated. A low post-thaw vitality in the meaning ofthe present invention means that not more than 50%, preferably not morethan 30%, more preferably not more than 20%, and furthermore preferablynot more than 15% of the cells are Annexin V positive within 6 h postthaw. In other words, a high quality cell bank is characterized in avitality-rate post thawing of more than 50%, preferably more than 70%,more preferably more than 80%, and furthermore preferably more than 85%,which means Annexin V negative within 6 h post thaw.

The term “long term preservation quality” is defined by the change ofthe three above described parameters over a prolonged period ofcryopreservation, for example about up to 20 years, of a given cellbank.

The necessity to remove serum from mammalian cell culture processes,particularly from culturing CHO cells, has resulted in highersensitivity of cultures towards cell death at different stages of aproduction process. A detailed analysis of cell death during thawing ofCHO cells subsequent to cryopreservation was provided. It was asurprisingly finding of the present invention that programmed cell deathor apoptosis is the predominant form of cell death caused bycryopreservation of CHO cells. As early as 3 h hours after, an increasedlevel of phosphatidylserine was surprisingly observed on the cellsurface of CHO cells. Display of phosphatidylserine on the surface ofCHO cells as a result from loss of phospholipids asymmetry is an earlyhallmark of programmed cell death. At this point, cellular membraneintegrity is still intact.

Early time points after thaw, e.g., 3 h to 24 h post thawing, showedAnnexin V positive stained CHO cells to still be morphologically intact(as seen in forward-side-scatter plots). At later time points (around24-48 h post thaw), the majority of Annexin V positive stained CHO cellsappeared to show signs of shrinking/membrane disassembly as seen byforward-side-scatter analysis. In accordance with these surprisinglyfindings, the percentage of CHO cells that could be stained withpropidium iodide (PI) only increased significantly 24 h post thaw. Thesedata describe for the first time nature and time course of cell death ofCHO cells post cryopreservation. Certain CHO cells show signs of havinginitiated a destruction program within a few hours after being placed inwarm culture medium and subsequently these cells disassemble over theperiod of 48 h.

Annexin V was initially described as a vascular protein with stronganticoagulant properties (Reutelsberger et al., 1985). It appears tobelong to a multigene family of proteins defined by a repeated motif,originally termed the endonexin loop. In the meantime, gene sequencescoding for the Annexin V protein of several species have been identifiedand cloned. For example, see NCBI Protein Data Base Accession No.NP_(—)01145.

The present invention therefore relates to the use of Annexin V incharacterizing a cryopreserved CHO cell bank. Particularly, the presentinvention relates to the use of Annexin V in a process of characterizinga cryopreserved cell bank of CHO cells. According to a furtherembodiment of the present invention, said process includes the steps: a)thawing a portion of cells of a cryopreserved cell bank; b) cultivatingsaid cells in a culture medium; c) incubating said cells with Annexin V;d) detecting cells and quantifying the number of cells that binds toAnnexin V; e) detecting cells and quantifying the number of cells thatdo not bind to Annexin V; and f) calculating the ratio of AnnexinV-binding versus Annexin V-non-binding cells.

According to a further embodiment the present invention also pertains tothe use of Annexin V in a process of determining the quality of a CHOcell bank, wherein said process includes the steps: a) cryopreservingcells into parts in a liquid medium as a cell bank; b) thawing a portionof the cryopreserved cells of said cell bank; c) cultivating said cellsin a culture medium; and d) establishing the vitality-rate of saidthawed cells by staining said cells with Annexin V. Criteria for thequality of a CHO cell bank are post-thaw vitality, robustness,phenotypic/genetic stability, and long term preservation quality,observed when cells are propagated and expanded starting from this cellbank. These criteria are mainly affected by the number of CHO cellswhich survive the freeze/thaw process, in other words the number of“intact cells.” A high number of intact CHO cells guarantee a fastgrowth of the initial cell culture, reduce the number of sub-cultivationsteps necessary to expand the cell culture up to the large fermentationscale and therefore to minimize the risk that genetic variants may beestablished and/or dominated the culture. The vitality-rate ispreferably estimated by Annexin V staining, which means the detectionand quantification of Annexin V-binding cells. Therefore, according to amore preferred embodiment of the present invention, the process of theAnnexin V staining in order to estimate the vitality-rate of CHO cellspropagated and expanded after thawing includes the steps: a) incubatingthe post-thawed cells with Annexin V; b) detecting cells and quantifyingthe number of cells that binds to Annexin V; c) detecting cells andquantifying the number of cells that do not bind to Annexin V; and d)calculating the ratio of Annexin V binding vs. Annexin V non-bindingcells. Examples of CHO cells are given in Table 1.

TABLE 1 CHO cell lines CHO Cell line Order Number CHO ECACC No. 8505302CHO-K1 ATCC CCL-61 CHO-DUKX ATCC CRL-9096 (=CHO duk-, CHO/dhfr-)CHO-DUKX B1 ATCC CRL-9010 CHO-DG44 Urlaub et al., Cell 33[2], 405-412,1983 CHO Pro-5 ATCC CRL-1781

A variety of assays for detection of apoptotic cell cultures has beendescribed, including DNA-laddering and TUNEL assays (Gavrieli et al., etal., 1992; Wijsman et al., 1993). However, flow cytometric methods ofapoptosis detection offer several advantages over the above techniquesas they allow rapid quantification of properties of thousands of cells.The use of flow-cytometric measurements allow rapid high throughputevaluation of different strategies do generate a successful cell bankfor a given cell line. For this reason and according to a furtherembodiment of the present invention, the Annexin V is labeled withfluorescein isothiocyanate (FITC). This allows the use of aflow-cytometric Annexin V affinity assays to estimate the vitality-rateof cells post thawing. Therefore, the present invention also pertains toa process of staining cryopreserved cells of a CHO cell bank postthawing a portion of cells of that cell bank with FITC-labeled Annexin Vin a flow-cytometric assay. An example of such an assay is given hereinmore in detail in the section Examples.

The results provided herein gives an explanation for results typicallyseen when a freeze/thaw step is monitored by trypan blue exclusionanalysis of cell viability (standard method which reflect the state ofthe art in characterization of cell banks). These classic thaw-controlexperiments generally show a high number of viable cells right afterthaw and viability only decreases between 24 h and 48 h post thaw. Abroad analysis of data obtained for CHO cell banks surprisingly revealedthe predictive value of flow-cytometric Annexin V affinity assays. Thepercentage of apoptotic CHO cells measured as early as 6 h post thawprovides information on the success of a cryopreservation strategy thatis usually only gained in classic thaw control experiments over a periodof 2-10 days. The present invention therefore also provide the use ofAnnexin V in characterization of a CHO cell bank or in a process ofdetermining the quality of a CHO cell bank, wherein the incubation stepwith Annexin V is performed within 24 h, preferably, 18 h, morepreferred 12 h, further more preferred 9 h, further more preferred 6 hpost-thaw and start of cultivation of the cryopreserved cells. In thisconnection use of flow-cytometric assay using FITC labeled Annexin V isthe most preferred method.

According to a further embodiment, the present invention also pertainsto the use of a Kit comprising Annexin V for the characterization of acryopreserved CHO cell bank post thawing, wherein Annexin V is used todetermine the vitality-rate of the cryopreserved CHO cells post thawing.According to a preferred embodiment of the present invention, thevitality-rate of a cell culture is determined by a process including thesteps: a) thawing a portion of cells of a cryopreserved cell bank; b)cultivating said cells in a culture medium; c) incubating said cellswith Annexin V; d) detecting cells and quantifying the number of cellsthat binds to Annexin V; e) detecting cells and quantifying the numberof cells that do not bind to Annexin V; and f) calculating the ratio ofAnnexin V-binding vs. Annexin V-non-binding cells. Furthermore, thepresent invention also provides a Kit comprising Annexin V and a packageleaflet including the information to use Annexin V in characterizationof a CHO cell bank.

The present invention further provides a process of characterizing a CHOcell bank including the steps: a) thawing a portion of cells of acryopreserved cell bank; b) cultivating said cells in an appropriateculture medium; c) incubating said cells with Annexin V; d) detectingcells and quantifying the number of cells that binds to Annexin V; e)detecting cells and quantifying the number of cells that do not bind toAnnexin V; and f) calculating the ratio of Annexin V-binding versusAnnexin V-non-binding cells. According to a further embodiment, thepresent invention also provides a process of measuring the vitality-rateof a CHO cell bank post-thaw, including the steps: a) thawing a portionof cells of a cryopreserved cell bank b) cultivating said cells in anappropriate culture medium c) incubating said cells with Annexin V; d)detecting cells and quantifying the number of cells that binds toAnnexin V; e) detecting cells and quantifying the number of cells thatdo not bind to Annexin V; and f) calculating the ratio of AnnexinV-binding vs. Annexin V-non-binding cells. As shown by the presentinvention, the percentage of apoptotic CHO cells measured as early as 24to 6 h post thaw provides information on the success of acryopreservation strategy for CHO cells that is usually only gained inclassic thaw control experiments over a period of 2-10 days. The presentinvention therefore also provide the processes of characterizing a CHOcell bank or measuring the vitality-rate of a cell bank post-thaw,wherein the incubation step with Annexin V is performed within 6-24hours post-thaw and start of cultivation of the cryopreserved cells.

CHO cells can be successfully stored in liquid nitrogen at around −196°C. for prolonged periods of time. However, cells can usually not befrozen and thawed without the addition of cryoprotectants such as DMSO.Even then, the freeze-thaw process will result in the loss of cells. Acritical parameter for the amount of this loss is the protocol used forthe freeze and the thaw process. Generally it is beneficial to controlthe temperature decrease during freezing to ensure that the temperaturedrops at a rate to of around one degree per minute. To thaw CHO cellssuccessfully it is required to warm the cryovial (thawed cells) as fastas possible to 37° C., the optimal cultivation temperature of almost allCHO cell lines. One way to ensure high recovery rates of cultures postthaw is to freeze cells in a medium containing high amounts of serum.

Recently, much work was focused on developing cryopreservationstrategies for CHO cells without the use of serum in the freezingmedium. A variety of cryoprotectants can facilitate successful long-termstorage of cells without the presence of serum. In addition, thecomposition of the basal medium as such has a significant impact on thesuccess of any cryopreservation. The described fast-track qualityassessment of any such strategy will allow drastically reduceddevelopment times that are needed to generate reliable protocols forgeneration of a master or working cell bank for new CHO production celllines. According to a further embodiment, the present invention alsoprovides a process of analysing a culture medium for CHO cells withregard to the use of said medium for cryopreservation of CHO cellsincluding the steps: a) cryopreserving CHO cells in a culture mediumsuitable for culturing those cells, preferably at temperatures below−100° C., e.g., in fluid nitrogen, b) thawing a portion or all of thecryopreserved cells; c) cultivating said cells in an appropriate culturemedium; d) incubating said cells with Annexin V; e) detecting cells andquantifying the number of cells that binds to Annexin V; f) detectingcells and quantifying the number of cells that do not bind to Annexin V;and g) calculating the ratio of Annexin V-binding versus AnnexinV-non-binding cells. Suitable culture media are those based oncommercially available media such as Ham's F12 (Sigma, Deisenhofen,Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM;Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's ModifiedDulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Calif.),CHO-S-Invtirogen), serum-free CHO Medium (Sigma), and protein-free CHOMedium (Sigma). According to a preferred process said medium includesspecific cryprotectants such as DMSO, BSA, methylcellulose, or glycine.According to a more preferred embodiment of this process, the incubationstep with Annexin V is performed within 6-24 hours post-thaw and startof cultivation of the cryopreserved cells. Use of FITC-labeled Annexin Vin flow-cytometric assay is a furthermore preferred embodiment of theinventive process.

The invention generally described above will be more readily understoodby reference to the following examples, which are hereby included merelyfor the purpose of illustration of certain embodiments of the presentinvention and are not intended to limit the invention in any way.

Abbreviations:

BSA: bovine serum albuminCHO: Chinese hamster ovaryDMSO: dimethyl sulfoxideELISA: enzyme-linked immunosorbant assayFACS: fluorescence activated cell sorterFITC: fluorescein isothiocyanateMCB: master cell bankPBS: phosphate buffered salinePCR: polymerase chain reactionPI: propidium iodidePPCB: post production cell bankPS: phosphatidylserineWCB: working cell bank

Methods: Cell Culture:

Cell banks of several CHO suspension cell lines used for production anddevelopment of biopharmaceuticals were investigated. All cell lines usedare proprietary and their protein product may not be revealed. All celllines used at production and development scale were maintained in serialseedstock cultures in surface-aerated T-Flasks (Nunc, Denmark) inincubators (Thermo, Germany) or sparged spinner flasks (Wheaton, USA) inspecially designed incubator rooms at a temperature of 37° C. and anappropriate mixture of air and 5% CO₂. Seedstock cultures were splitevery 2-3 days with an appropriate splitting ratio and seed density.Cell concentration was determined in all cultures by using ahemocytometer. Viability was assessed by the trypan blue exclusionmethod. The cultures originated from master, working or safety cellbanks and were thoroughly tested for sterility, mycoplasma, and thepresence of adventitious agents. All operations took place inair-filtered laboratories and under strict procedures complying with“current Good Manufacturing Practices (cGMP). All culture media used areproprietary and their composition may not be revealed.

Cryopreservation:

Freezing and thawing were performed according to standard protocols andthe fundamental law “freezing low-thawing fast.” The cultures were takenin exponential phase from suspension cultures and were frozen in 1.8 mLplastic vials (Nunc, Demark) either by a computer-controlled freezingmachine (Consarctic, Germany) with a specially designed freezing programor by a Styrofoam box in a −70° C. freezer. After freezing to −100° C.(freezing machine) or −70° C. (Styrofoam box) the cells were transferredto liquid nitrogen containers. Vials were stored in the gas phase of theliquid nitrogen (<−150° C.). The cell concentration in the freezingvials was 1-3E7 cells. DMSO (Merck, Germany) was used as acryoprotectant at a concentration of 10%. The detailed freezing mediaused are also proprietary and their composition may not be revealed. CHOcells were thawed in an 37° C. water bath, diluted with appropriatemedium, and centrifuged (Thermo, France) at 180×g. Subsequently cellswere seeded in cultivation medium with a defined cell concentration.

Flow Cytometry:

Green Annexin-V-FITC and red PI fluorescence were measured with aCoulter Epics XL flow cytometer (Coulter, Germany) using Expo32software. Excitation was elicited at 488 nm with an Argon laser andmeasured using the standard band pass (530±20 nm) and long pass (>570nm) filters. In each sample, 10000 events for apoptosis and 3000 eventsfor cell cycle analysis were measured. Data were analyzed with theExpo32 analysis tool. To discriminate between dead and apoptotic cells,the membrane-impermeable DNA stain propidium iodide (PI) was added inparallel to Annexin V to the cell suspension. With this double stainingit is possible to discriminate between vital, early-apoptotic,late-apoptotic, and necrotic cells. The Annexin V assay was performedaccording to the vendor's protocol (Becton-Dickinson, Germany). Thecells were analyzed and apoptosis was quantified by flow cytometry.Propidium iodide staining was further used to determine cell cycledistribution. Samples were taken from cell suspensions without fixation.Cultures were centrifuged and washed with PBS (Gibco, Germany). Then,the PI staining solution (BioSure, USA) was added and incubationproceeded for 30 min. Subsequently, cells were analyzed by flowcytometry. The proportion of cells in each phase of the cell cycle wasobtained through the Multicycle program (Phoenix Flow, USA).

Results:

Quantitative analysis of cell death during the thaw of cryopreserved CHOcells:

Although it is known that CHO cells die when cultures of cells arereinitiated after cryopreservation the kinetic and nature of this celldeath have not been described in great detail. To quantify the extent ofprogrammed cell death in cell cultures of CHO cells during the firsthours after thawing we determined the amount of phosphatidylserinedisplayed on the outer membrane of CHO cells. CHO DG44 cells andderivatives of this cell line are widely used for production ofbiopharmaceuticals. We first analyzed a cell bank of 220 vials generatedfor a CHO-derived production cell line. The cell bank was generated witha freezing medium free of animal components, such as serum, and wasknown to be particularly sensitive with regard to culture viabilityafter thawing. Cells were analyzed by double staining with Annexin V andpropidium iodide at time points Oh, 3 h, 6 h, 24 h, and 48 h. A highpercentage of approximately 30% Annexin V positive cells was detectedright after cells were placed in cultivation medium (Oh), increasing toalmost 50% within the first 3 h, and remaining at above 40% for thefirst 48 h post thaw. For all CHO DG44 cell banks analyzed so far, thepeak level of Annexin V positive cells was reached between 3 h and 24 hpost thaw. In contrast to that, the amount of PI positive cells remainedrelatively low immediately after thaw before it increased markedly 24 hpost thaw. In the initial phase of apoptosis, PS is displayed on thecell surface and Annexin V binds it target. In this early phase, theplasma membrane is still able to exclude PI and, thus, cells are AnnexinV+/PI− (FIG. 1, lower right quadrant). Subsequently, cells loose theirplasma membrane integrity and their ability to exclude PI. These lateapoptotic cells are Annexin V+/PI+ (FIG. 1, upper right quadrant).Therefore, these experiments demonstrate that the transition from earlyto late stage apoptosis only occurs after the first 6 hours post thaw.This is also seen in the forward-side scatter diagram (FIG. 1), showingan increase in very small cells, representing destructed cell bodies(FIG. 1) at the transition point around 24 hours post thaw.

Post-Thaw Analysis by Annexin V Staining Serves as Predictive EarlyMarker for CHO Cell Bank Quality:

As quantification of apoptosis by Annexin V staining can be performedrapidly for a high number of samples it offers an attractive method forhigh-throughput analysis of different cryopreservation methods. Weanalyzed two CHO cell banks of two different CHO production cells byAnnexin V staining and compared these data to trypan blue exclusionstaining measured for six passages in culture (standard thaw control).As seen in FIG. 2A, the amount of phosphatidylserine-displaying cellsvaried significantly between cultures thawed from the two different cellbanks (35% in thaw 1 versus 67% in thaw 2). The classic thawing control(FIG. 2B) showed the same significant difference between the cultureviabilities from the different banks. Thaw 1 demonstrated a drop to 83%before the culture recovered to a viability of more than 90% four dayspost thaw. In contrast to that, only 60% of cells were viable 24 h afterthaw 2. This culture took eleven days to reach 90% viability asdetermined by trypan blue exclusion. A cell bank of this quality wouldclearly not be suitable for a commercial production process. These datasurprisingly demonstrate that the percentage of cells displaying PS ontheir surface predicts the outcome of classic thawing controls as earlyas 6 h post thaw.

Cell Bank Quality can Vary Dramatically Amongst Different Cell Lines,Cryopreservation Protocols, and Quality of Cultivation Medium:

To test the newly described method for cell bank quality assessment weanalyzed a number of cell banks. Two CHO DG44 cell banks were generated,one containing 0.8% BSA in the freezing medium, the other containing a50:50 mixture of conditioned and fresh medium without BSA. As depictedin FIG. 3, the cells showed significantly lower rates of early apoptosisfor both freezing media. Furthermore, a beneficial effect of adding 0.8%BSA compared to storing cells in conditioned medium was only evidentwhen styrofoam boxes were used.

Use of Early Post-Thaw Apoptosis Measurements for Fast-Track DevelopmentStrategies:

As time to clinic and market supply is a crucial parameter forsuccessful development of new biopharmaceuticals there is a growinginterest in fast-track screening methods for process optimization andprocess improvements. We therefore determined the feasibility of usingAnnexin V measurements just 6 h post thaw to evaluate strategies forestablishing successful cryopreservation of new CHO production celllines.

We surprisingly find out that early post-thaw apoptosis measurements area sensitive tool for assessment of raw materials quality. An essentialfactor for successful cryopreservation of CHO cells is the quality ofthe medium used. Generally, GMP includes clearly defined expiry datesfor use of media for culturing CHO cells. We investigated whether theharsh conditions of a freeze-thaw process may result in shortertimeframes for the use of media compared to standard cultivationprocedures. We used a medium with a shelf life of four weeks forcultivation of CHO cells to investigate this further. FIG. 4 shows earlyapoptosis data (6 h post thaw) for CHO cells that were cryopreserved infreshly prepared medium (4 bottom), medium that was stored at 4 C forfour weeks (4 top) and medium that was stored at 4 C for four weeks andwas supplemented with a fresh preparation of an essential compoundimmediately before use (4 middle). These data show a decreased thawperformance for cells banks generated with this medium just before itsexpiration date. This effect could be reduced significantly by adding anewly prepared essential component of the relevant medium. Simultaneousdetermination of the cell cycle distribution 6 h post thaw showed thatfour week old medium had no growth inhibitory effect. These resultssurprisingly demonstrate how early post-thaw apoptosis measurementscould be used for sensitive analyses of media and their components withregard to the use of media preparations for cryopreservation of CHOcells.

1-8. (canceled)
 9. A method for establishing the vitality rate of acryopreserved CHO cell bank, the method comprising staining thawed CHOcells from the cryopreserved CHO cell bank with Annexin V.
 10. Themethod of claim 9, further comprising: quantifying the number of thawedCHO cells that binds to Annexin V; quantifying the number of thawed CHOcells that do not bind to Annexin V; and calculating the ratio ofAnnexin V-binding thawed CHO cells to Annexin V-non-binding thawed CHOcells.
 11. The method of claim 10, wherein Annexin V is incubated withthe thawed CHO cells within 24 hours post-thaw.
 12. The method of claim10, wherein Annexin V is incubated with the thawed CHO cells within 6hours post-thaw.
 13. The method of claim 10, wherein the Annexin V islabeled with fluorescein isothiocyanate (FITC).
 14. The method of claim10, wherein the cryopreserved cell bank of CHO cells comprises CHO cellsthat are cryopreserved into parts in a liquid medium.
 15. The method ofclaim 10, wherein the thawed CHO cells are thawed from a portion of thecryopreserved cell bank.
 16. The method of claim 10, wherein the thawedCHO cells are cultured before being incubated with Annexin V. 17.(canceled)
 18. (canceled)